PROJECT SUMMARY Metastatic colorectal cancer (CRC) is an aggressive disease impacting about 50,000 deaths annually in the USA. Patients with metastatic CRC are predominantly unresponsive to existing therapies. The metastatic process is mediated in part by dysregulated translation of oncogenic mRNAs, leading to overproduction of their encoded proteins. Previous findings established dysregulation of cap-dependent mRNA translation downstream of mTOR at the level of 4E-BP1/eIF4E as a key to tumor formation and metastatic progression in CRC. While targeting mTOR is thought to be a promising strategy for CRC therapy, limited therapeutic efficacy of mTOR inhibitor drugs correlates largely with loss of the translation repressive function of 4E-BP1. More recent findings indicate that Snail acts as a strong repressor of 4E-BP1 transcription and cooperates with mTOR-mediated phosphorylation (inactivation) of 4E-BP1 to significantly increase eIF4E-initiated cap-dependent mRNA translation. These processes support tumor growth and decrease the efficacy of the mTOR kinase (ATP- competitive) inhibitors (mTORkis) in CRC therapy. Although mTORkis effectively inhibit phosphorylation of 4E- BP1 and restore its repressive effects on cap-dependent translation and tumor growth, treatment with mTORkis in CRC cells can promote the active translation and expression of the immunosuppressive protein PD-L1 via initiation at an internal ribosome entry site (IRES) in a cap-independent manner. In addition, the RNA helicase eIF4A is a key PD-L1 IRES binding protein that regulates its translation and expression. Importantly, elevated PD-L1 levels induced by mTORkis result in evasion of anti-CRC immunity. Further, targeted inhibition of PD-L1 can restore T-cell immunity and enhance the efficacy of mTORkis. Based on these findings, the central hypothesis of the proposed study is that CRC cells usurp the regulatory mechanisms underlying both cap- dependent translation through co-activation of Snail and mTOR and IRES-mediated translation of PD-L1 to escape immune surveillance in mTOR kinase-targeted therapy, thereby causing CRC resistance to mTORkis and promoting CRC progression. To test this hypothesis, the following specific aims are proposed: 1) to identify how Snail cooperates with mTOR in translational control of CRC progression and modulation of mTOR kinase- targeted therapy; 2) to determine the cap-independent mechanism of PD-L1 mRNA translation upon mTOR kinase inhibition; and 3) to define the in vivo utility of co-targeting PD-L1 and mTOR to enhance CRC therapy. The focus of this study is the innovative concept that both Snail and PD-L1 promote CRC progression by cooperating with mTOR to modulate therapeutic response to mTORkis through dysregulation of 4E-BP1- mediated translation initiation processes. This research will not only define the novel mechanistic roles of both Snail and PD-L1 in the modulation of mTOR/4E-BP1-mediated translational control of CRC progression and resistance to mTORkis, but also facilitate rational approaches for the development of new translatable therapeutic strategies for patients with advanced CRC.